Autophagy is a catabolic cellular process by which double membranous vacuoles engulf and degrade cytoplasmic material. This constitutively active mechanism is crucial for the intracellular housekeeping of the cell. However, under stress conditions such as hypoxia, nutrient deprivation or DNA damage, autophagy is highly activated to restore energy production and promote survival. Activation of autophagy is therefore one of the strategies used by tumors in response to hypoxia and chemotherapy allowing it to survive the harsh microenvironment and to evade cell death. Indeed, pronounced accumulation of the autophagy marker microtubule-associated protein 1 (LC3B) is often predictive of poor prognosis in cancer patients. Following multiple preclinical studies reporting beneficial effects in tumor models upon autophagy inhibition, clinical trials featuring hydroxychloroquine were initiated to enhance the effectiveness of chemotherapy in patients. Hydroxychloroquine is a non-specific late autophagy inhibitor which abrogates the autophagosome-lysosome fusion, the last step of the autophagic flux. However, the drug is highly unspecific and can cause toxicity. A critical component of the autophagy machinery is the cysteine protease Atg4 (autophagy-related gene-4). Among the four distinct forms identified in mammals, only Atg4B has been shown to have an important role in autophagy (Li et al., 2011). The enzyme converts pro-LC3B to LC3B-I by cleaving its C-terminus after which LC3B-I can be conjugated to phosphatidylethanolamine by other Atgs, thereby forming LC3B-II. This lipidation process of LC3B-I allows expansion and closure of autophagosomes. Given the encouraging results following Atg4B inhibition in several tumors (Akin et al., 2014; Apel et al., 2008; Rothe et al., 2014), the enzyme is becoming increasingly attractive as a therapeutic target for cancer and considerable efforts have recently been made to identify small molecule Atg4B-inhibitors (Ketteler and Seed, 2008; Shu et al., 2010, 2011).
In the current invention, we disclose the discovery of novel autophagy inhibitors with a benzotropolone core structure. The benzotropolone scaffold is present in numerous known synthetic compounds and natural products and potential applications of benzotropolone-containing molecules are described in diverse fields. Examples include use as food stabilizers, antimicrobial agents, sunscreens, cosmetics and anti-obesity agents. However, this class of compounds was never investigated for autophagy modulation. In contrast, one benzotropolone was reported as a weak autophagy inhibitor by Shu et al. and was even reported as unattractive as a starting point for the development of potent and selective autophagy inhibitors. Furthermore, this compound showed no in cellulo autophagy inhibition. Moreover, in our hands, this compound did not show activity in a cellular screening assay for autophagy inhibitors.

Therefore, surprisingly we have found potent in cellulo autophagy inhibition for a well-defined series of novel analogues of the reported molecule, which are characterized by having a halogen atom as specified in the claims. Furthermore, these halogenated analogues not only showed promising activity in living cells but also showed an improved stability in blood plasma and in the presence of liver microsomes.
While, the synthesis of one such halogenated benzotropolone compound has already been described in the early '60s (DE1091114, example 16), this type of compounds has not been further explored in the meantime, and its public disclosure is only limited to the synthesis route thereof, while no utility is described for that particular compound, or more generally the group of halogenated benzotropolones.
